Advancements in the construction of transgenic mice bearing human antibody gene loci have led researchers to move away from the provision of entirely human transgenic antibody loci (bearing human variable and constant region gene segments) to chimaeric transgenic antibody loci. The transgenic loci comprise human V(D)J segments operably connected upstream of non-human constant regions. The use of constant regions that are endogenous to the transgenic non-human vertebrate (eg, endogenous mouse or rat constant regions) are desirable to harness the endogenous control of antibody generation and maturation following immunisation of the vertebrate with antigen.
B-cell development is characterized by the ordered rearrangement of immunoglobulin variable region genes. After the VDJ rearrangement of the H chain gene segments, a precursor (pre)-B-cell is generated. After the functional rearrangement of a light chain gene, the cells develop into surface IgM-bearing mature B-cells. Fully assembled IgH and L chains are transported onto the surface of B-cells, while free H chains are retained in the endoplasmic reticulum (ER) in association with BiP.
A critical step in B-cell differentiation is the selective expansion of cells with a functional μ heavy chain resulting from productive IgH rearrangement. This is achieved by the association of the μ heavy chain with the surrogate light chain (SLC) proteins λ5 and VpreB and the signal transducing heterodimer Igα and β to form the pre-B-cell receptor (pre-BCR). The expression and formation of a pre-BCR dramatically improves the efficiency of pre-B and B-cell production, by signalling proliferative expansion of pre-B-cells. The major function of the pre-BCR is the selection and expansion of cells that have undergone a productive VDJ rearrangement. The expression of membrane-bound μ chains is essential for the clonal expansion, and initiation of L chain gene rearrangement.
The association of SLC with μ chain works as a checkpoint to determine whether the cell has successfully completed VDJ combination and expresses a functional μH chain. More than half of all μ chains cannot assemble with the SLC, accounting for most of the changes in the V heavy repertoire during B-cell development.
VpreB is homologous to variable regions, λ 5 is homologous to light chain constant region. The protein encoded by the two genes can form a tightly, but noncovalently bound heterodimer with the general structure of an L chain. λ 5 protein can be covalently disulphide-bonded to μ H chains in pre-B-cells. Whereas VpreB alone can associate with Igμ, λ 5 alone cannot. Only when it is non-covalently associated with VpreB does λ 5 form a disulphide bridge with the first constant region (CH1) of an Igμ chain to form the pre-BCR.
The λ5 and VpreB, which together form the SLC, are early markers of B-cell commitment. They are expressed at the pro- and pre-B-cell stages and silenced in immature and mature B-cells. In pre-B-cells, following rearrangement of the heavy chain locus, the surrogate light chain (SLC) acts as a chaperone, mediating transport of the newly synthesised heavy chain mu to the cell surface and together with C mu forms part of the pre-BCR (Immuno) Today. 1993 February; 14(2):60-8; The surrogate light chain in B-cell development; Melchers F, Karasuyama H, Haasner D, Bauer S, Kudo A, Sakaguchi N, Jameson B, Rolink A). The pre-BCR mediates signalling, leading to proliferation of pre-B-cells that have a productive heavy chain rearrangement. Mice that lack a functional λ5 show a drastic reduction in the number of B-cells. Using VpreB knock-out mice, it has been shown that VpreB is also required for efficient B-cell development, particularly for the transition to pre-BCR bearing cells (pre-BII stage) (see, Int Immunol. 1999 March; 11(3):453-60; Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice; M{dot over (a)}rtensson A, Argon Y, Melchers F, Dul J L, M{dot over (a)}rtensson I L; and Sabbattini & Dillon 2005 infra). As the degree of proliferation has been proposed to be dependent upon the stability of the association of the mu heavy chain with the components of the SLC, the pre-BCR is thought to influence the choice, and thus the extent and diversity of, the heavy chain variable region repertoire.
Besides its role in signalling proliferation, the pre-BCR is also thought to mediate down regulation of the RAG genes, thereby preventing the rearrangement of other IgH loci (allelic exclusion) and the occurrence of double-strand breaks in dividing pre-B-cells. The pre-BCR has also been proposed to exert a negative feedback on λ5 and VpreB expression so that, after a phase of clonal expansion, the large pre-BII cells become depleted of pre-BCR, exit the cell cycle and differentiate into resting small pre-BII cells. At this stage, the SLC is repressed while the IgL loci start to be rearranged. The product of a successfully rearranged IgL gene will then pair with the mu heavy chain to form a BCR with antigen-binding capability on the surface of immature B-cells. These cells migrate to the peripheral blood and secondary lymphoid organs, and develop into mature B-cells ready for subsequent encounter with antigen (Sabbattini & Dillon 2005 infra).